[Epilepsy in cancer patients: primary prevention and the importance of high-risk patient screening]. / Epilepsia en el paciente oncológico: prevención primaria e importancia en la selección del paciente de alto riesgo.

INTRODUCTION: Epilepsy in cancer patients has a prevalence of 13%, and is especially high in patients with brain tumours, with a higher morbidity and mortality rate compared to non-tumour-related epilepsy. Its physiopathogenic mechanisms are distinct and include distortion of the cortical architecture and alteration of the glutamate-enhancing tumoural and peritumoural molecular microenvironment. Nevertheless, there is scarce and inconsistent scientific evidence on some fundamental aspects, such as primary post-operative prophylaxis, the ideal pharmacological profile or the withdrawal time of antiseizure drugs after their release. DEVELOPMENT: Characteristics such as low tumour grade, number/size of cortical lesions, location (frontal, cortical/subcortical or eloquent area), early seizures and molecular alterations, such as IDH1/2 mutation, are factors that favour the occurrence of seizures. Within the treatment, surgery will provide cytoreduction and seizure control by excision of the epileptogenic area, with 75-90% freedom from disabling seizures. Although still a controversial issue, the post-operative use of antiseizure drugs is contraindicated by the main scientific societies due to the scarce evidence and the wide spectrum of side effects. However, they are frequently used in daily clinical practice. CONCLUSIONS: All this forces us to establish a group of patients at 'high risk' of postoperative seizures, who will need to select the ideal antiseizure drug for primary prevention, with a route of administration that facilitates a rapid action effect and pharmacokinetics that prevents hepatic metabolism and CYP450 induction to achieve a lower number of interactions with chemotherapy, corticosteroids and radiotherapy. Despite this, drug resistance rates of 20-40% and relapse rates of 25-29% have been reported.

TITLE: Epilepsia en el paciente oncológico: prevención primaria e importancia en la selección del paciente de alto riesgo.Introducción. La epilepsia en el paciente oncológico presenta una prevalencia del 13%, especialmente elevada en pacientes con tumores cerebrales, así como una mayor morbimortalidad respecto de la epilepsia no tumoral. Sus mecanismos fisiopatógenos son diferenciadores, e incluyen la distorsión de la arquitectura cortical y la alteración del microambiente molecular tumoral y peritumoral favorecedor de glutamato. A pesar de ello, existe evidencia científica escasa e inconsistente acerca de aspectos fundamentales, como la profilaxis primaria postoperatoria, el perfil farmacológico idóneo o el tiempo de retirada de fármacos anticrisis tras la libertad de éstas. Desarrollo. Características como el bajo grado tumoral, el número/tamaño de las lesiones corticales, la localización (frontal, cortical/subcortical o área elocuente), las crisis tempranas y las alteraciones moleculares, como mutación IDH1/2, son factores favorecedores para la aparición de crisis. Dentro del tratamiento, la cirugía aportará citorreducción y control de crisis por escisión del área epileptógena, con libertad de crisis incapacitantes del 75-90%. Aunque sigue siendo un tema controvertido, el uso postoperatorio de fármacos anticrisis está contraindicado por las principales sociedades científicas por la escasa evidencia y el amplio espectro de efectos secundarios. Sin embargo, se emplean frecuentemente en la práctica clínica diaria. Conclusiones. Todo ello nos obliga a establecer un grupo de pacientes de 'alto riesgo' de crisis postoperatorias, que precisará seleccionar el fármaco anticrisis idóneo en prevención primaria, con una vía de administración que facilite un rápido efecto de acción y una farmacocinética que evite el metabolismo hepático y la inducción de CYP450 para conseguir un menor número de interacciones con quimioterápicos, corticoides y radioterapia. A pesar de ello, se describen tasas de farmacorresistencia del 20-40% y recidiva del 25-29%.

Glucagon-stimulable adenylyl cyclase in rat liver. Effects of chronic uremia and intermittent glucagon administration.

The effects of chronic uremia and glucagon administration on glucagon-stimulable adenylyl cyclase in rat liver were assessed by determinations of adenylyl cyclase activities, specific iodoglucagon binding, and the activity of the stimulatory regulatory component of adenylyl cyclase. Glucagon-stimulated adenylyl cyclase was reduced in uremia to 75-80% of control levels (P less than 0.05), in the presence or absence of saturating levels of guanosine triphosphate (GTP) and 5'-guanylylimidodiphosphate [GMP-P(NH)P]. Although these changes were accompanied by a concomitant 20% reduction in sodium fluoride-stimulated activity, basal, GTP-, GMP-P(NH)P-, and manganese-dependent adenylyl cyclase activities were unchanged. Using [125I-Tyr10]monoiodoglucagon as a receptor probe, the number of high affinity glucagon-binding sites was reduced 28% (P less than 0.01) in uremic as compared with control liver membranes. However, the affinity of these binding sites was unaltered. The S49 cyc- -reconstituting activity with respect to both GMP-P(NH)P- and isoproterenol plus GTP-stimulable adenylyl cyclase was unaltered in membranes from uremic as compared with control rats. Intermittent glucagon (80-100 micrograms) injections administered at 8-h intervals to normal rats reproduced all of the above described effects of chronic experimental uremia on the adenylyl cyclase system. It is concluded that changes in the hormone-stimulable adenylyl cyclase complex in uremia and with glucagon treatment result primarily from a decrease in the number of hormone-specific receptor sites in hepatic plasma membranes. Since the changes in liver adenylyl cyclase are qualitatively and quantitatively the same in glucagon-treated and uremic rats, it is suggested that these may be the result of the hyperglucagonemia of uremia. Further, the data reveal an unexpected dissociation between guanine nucleotide and sodium fluoride stimulation of adenylyl cyclase. Possible causes for this dissociation based on the known subunit composition of cyclase coupling proteins are discussed.

Glucagon-stimulable adenylyl cyclase in rat liver. The impact of streptozotocin-induced diabetes mellitus.

Glucagon receptor levels, glucagon-stimulated and other forms of adenylyl cyclase activity, and regulatory component activity of adenylyl cyclase were determined in hepatic plasma membranes of rats administered streptozotocin without and with insulin to produce varying degrees of hyperglycemia. Receptor levels were assayed by direct binding of the specific probe [125I-Tyr10]-iodoglucagon; regulatory component activity was assayed by the capacity to reconstitute stimulatory regulation in deficient membranes from cyc- S49 murine lymphoma cells. In rats given 150 mg streptozotocin, glucagon stimulation of adenylyl cyclase as well as basal, sodium fluoride, 5' guanylylimidodiphosphate [GMP-P(NH)P] and Mn-dependent activities were reduced 50%, glucagon receptor levels but not affinity were reduced 67%, and regulatory component activity was decreased 50%. In addition, alpha 1-adrenergic receptors and 5'-nucleotidase were similarly reduced in diabetes. However, specific ouabain-inhibitable Na+, K+, ATPase activity was not altered by streptozotocin treatment. The streptozotocin-induced changes were noted within 24 h and became maximal by 120 h after its administration. All of these decreases were partially reversed by in vivo insulin treatment. DNA, cytochrome c oxidase, glucose-6-phosphatase, and N-acetyl-beta-glucosaminidase content in hepatic plasma membrane preparations were not substantially different in diabetic as compared with control animals. The data demonstrate that glucagon-mediated regulation of cyclic AMP formation is deranged in insulin deficiency owing to a combined decrease in receptors, derangement of the coupling mechanism intervening between receptor and adenylyl cyclase, and possibly, an altered basal effector system. Some of these changes appear to reflect a "desensitization-like" phenomenon which may or may not be attributable to the hyperglucagonemia of diabetes mellitus. There also appears to be a concurrent generalized decrease in several but not all plasma membrane receptor and enzymatic proteins. This may be the result of a number of processes among which is the accelerated proteolysis of uncontrolled diabetes.

Gonadal receptors. II. Effects of time and reaction volume upon the binding of human chorionic gonadotropin and human luteinizing hormone to particulate receptors.

The effect of reaction volume upon the binding of gonadotropins by particulate receptors was studied. Two experimental approaches were used: one involved increasing the reaction volume of the binding assay (i.e. diluting the hormone and receptor concentrations and will be referred to as buffer coincubation studies) and the other involved incubating the testicular homogenate in various buffer volumes prior to the binding assay (buffer preincubation stidies). The results showed that the number of hormone binding sites inferred from Scatchard analysis was inversely related to the reaction volume in the coincubation as well as in the preincubation studies. Time-dependent dissociation of receptors from the intact testis was demonstrated by perifusion studies and the loss of receptors from intact testis correlated with the appearance of soluble factors (Bhalla, V.K., Haskell, J., Grier, H. and Mahesh, V.B. (1976) J. Biol. Chem. 251, 4947--4957) in the eluate obtained. The results obtained along with those presented in the preceding manuscript (Chen, C.J.H., Lindeman, J.G., Trowbridge, C.G. and Bhalla, V.K. (1979) Biochim. Biophys. Acta 284, 407--435) question the validity of the rapid equilibrium model which assumes reversible hormone occupancy of a fixed number of receptor sites. An alternate binding model is proposed herein and its implications are discussed.

Evidence for a novel adenylyl cyclase in human epididymal sperm.

Since cAMP is considered to play a major role in the acquisition of maturation and fertilizing capacity of mammalian sperm, we investigated the expression of cAMP-synthesizing adenylyl cyclase (AC) in sperm retrieved directly from the human epididymis. Particulate fractions were prepared from purified epididymal sperm samples and AC was monitored by the direct conversion of ATP into cAMP. We report that in great contrast to human ejaculated sperm and other mammalian sperm cells, the human epididymal sperm do not express a Mn(2+)-sensitive AC. However, a functional AC was readily detectable in these sperm cells in the presence of saturating concentrations of Ca2+ (50mM) and bicarbonate (HCO3-, 50mM), a combination that causes maximal activation in human ejaculated sperm. Using these conditions, human epididymal sperm AC showed similar capacity to generate cAMP compared to human ejaculated sperm AC. When assays were performed in the presence of Mg2+ and a saturating concentration of GMP-P(NH)P (50 microM), the hydrolysis-resistant GTP analog, and forskolin (100 microM), no activity was detected indicating that the epididymal sperm AC differs from that in somatic cells. These data demonstrate that human epididymal sperm contain an AC that is unique and different from the enzyme system described in somatic cells and other mammalian sperm cells, including human ejaculated sperm.

Monoiodoglucagon: synthesis, purification by high pressure liquid chromatography, and characteristics as a receptor probe.

The synthesis of [125I-Tyr10]monoiodoglucagon from glucagon and carrier-free 125I using 1,3,4,6-tetrachloro-3-6-diphenylglycouril (Iodogen) and its separation in pure form by reverse phase high pressure liquid chromatography (HPLC) over C18-muBondapak columns using two consecutive linear gradients between solvent A [40:60 mixture of methanol and 10 mM H3PO4 in H2O (pH adjusted to 3.0 with triethylamine)] and solvent B (50:50 mixture of acetonitrile and 0.1 M Tris-HCl, pH 9.0) is reported. The newly synthesized [125I]monoiodoglucagon is shown to activate adenylyl cyclase in liver membranes with an EC50 between 5- and 8-fold lower than that of native glucagon. Further, it binds specifically to sites on liver plasma membranes that have the characteristics of glucagon receptors in terms of guanine nucleotide sensitivity and rates of reaction. It is suggested that [125I-Tyr10]monoiodoglucagon is a suitable probe for studying structural and functional properties of glucagon receptors.

Properties of gonadotropin-stimulable adenylyl cyclase of the human corpus luteum: regulation of hormonal responsiveness by guanyl nucleotide and magnesium ion.

We studied the properties of a gonadotropin-responsive adenylyl cyclase in membrane preparations obtained from human corpus luteum and explored the nature of guanyl nucleotide and Mg ion involvement in activation of the enzyme. Maximal adenylyl cyclase activity required ATP concentrations of 1-2.5 mM, total MgCl2 concentrations of 8-10 mM, and 1 mM EDTA. Optimal hCG responsiveness, however, required lower (approximately 5 mM) MgCl2 concentrations. Both GTP and its hydrolysis-resistant analog guanyl 5'-yl imidodiphosphate [GMP-P(NH)P] increased enzyme activity, but the response to each nucleotide had distinct characteristics. The rate and the extent of activation were greater in the presence of GMP-P(NH)P than in that of GTP. Moreover, enzyme activation by GMP-P(NH)P was hysteretic in nature, requiring about 8 min to reach steady state velocity in the absence of hormonal stimuli. The slow rate of activation by GMP-P(NH)P was accelerated by either hCG or increases (3-10 mM) in the concentration of MgCl2. Thus, both gonadotropin and Mg ion are inherently antihysteretic in the human luteal adenylyl cyclase system. Basal and hCG stimulation were under the control of guanine nucleotides. Dose-response curves showed that the apparent activation constants for GTP and GMP-P(NH)P were 0.30 and 0.51 microM, respectively; these values did not shift after the addition of hCG. At a higher concentration of guanyl nucleotides (1000 microM), basal and hCG-stimulated activities were markedly reduced, suggesting bimodal regulation of the enzyme by the nucleotides. We also found that enzyme responsiveness to prostaglandin E2 was small and that, in contrast to a number of other nonprimate species, adenylyl cyclase from the human corpus luteum was not stimulated by isoproterenol. Taken together, these data support the usefulness of the cell-free model for studying the role of adenylyl cyclase in the regulation of luteal function in the human.

Changes in adenylyl cyclase activity of the human and nonhuman primate corpus luteum during the menstrual cycle and pregnancy.

Adenylyl cyclase (AC) activity in membrane particles of corpora lutea (CL) from humans and cynomolgus monkeys was examined at various stages of the menstrual cycle and pregnancy. AC activity was monitored by the conversion of [alpha-32P]ATP into [32P]cAMP under basal conditions and in the presence of several activators: NaF (10 mmol/L) plus forskolin (100 mumol/L); hCG (10 micrograms/mL); guanyl 5'-yl-imidodiphosphate [GMP-P(NH)P; 100 mumol/L]; and hCG (10 micrograms/ml) plus GMP-P(NH)P (100 mumol/L). The groups of human CL were midluteal (n = 10), late luteal (n = 4), following cycle (old CL; n = 5), and early pregnancy (6-11 weeks; n = 10). The groups of monkey CL were early luteal (n = 4), midluteal (n = 5), and pregnancy at term (n = 3). Luteal AC activity changed significantly during the menstrual cycle. In newly (less than 48 h after ovulation) formed CL, the enzyme was unresponsive to hCG, and total AC activity, as determined by NaF plus forskolin, averaged 86.5 +/- 28.9 (+/- SE) pmol cAMP/min.mg protein. As the CL developed, AC activity increased. Thus, in the midluteal phase, maximal hCG responsiveness in the presence of guanine nucleotide was 125 +/- 27 and 232 +/- 15 pmol/min.mg in human and monkey CL, respectively. No hCG responsiveness was detected in the late luteal phase or in the old CL. Maximal AC activity was also high in the midluteal phase (382 +/- 56 and 256 +/- 28 pmol/min.mg in human and monkey CL, respectively); the activity remained fairly high during the late luteal phase and then declined to less than 100 pmol/min.mg in the follicular phase of the next cycle. During early pregnancy, luteal AC was unresponsive to hCG stimulation, yet basal levels, maximal activity, and the characteristics of stimulation by nonhormonal activators were similar, if not identical, to those at the midluteal phase of the menstrual cycle. At term pregnancy, the enzyme remained unresponsive to hCG. However, basal activity and stimulation by NaF and forskolin were remarkably elevated, being between 2- and 7-fold higher than corresponding stimulations in the midluteal phase. We conclude that 1) AC activity in human luteal membranes is highly dependent on hormonal changes and functional state of the ovary, 2) the activity of luteal AC is similar in the CL of humans and cynomolgus monkeys, and 3) the AC system in the primate CL is functionally active during and at the end of pregnancy.(ABSTRACT TRUNCATED AT 400 WORDS)

Inhibition of luteinizing hormone/human chorionic gonadotropin-stimulable adenylyl cyclase activity in rat luteal membranes by nonsteroidal component(s) in human follicular fluid.

We examined the ability of nonsteroidal components of human follicular fluid (hFF) to alter gonadotropin responsiveness using the LH/hCG-sensitive adenylyl cyclase system of rat luteal membranes. Follicular aspirates were obtained from regularly ovulatory women (n = 10) whose follicles were stimulated by human menopausal gonadotropin and hCG as part of an in vitro fertilization program. hFF from large follicles was pooled and extracted with 10% (wt/vol) activated charcoal. Maximal hCG stimulation of adenylyl cyclase activity obtained with 10 micrograms/ml hCG and 100 microM of the hydrolysis-resistant GTP analog guanyl 5'-yl-imidodiphosphate was significantly inhibited by hFF in a dose-dependent manner. Addition of about 500 micrograms hFF protein caused inhibition of 70% compared to the control value. Fractionations of hFF by ultrafiltration using membranes of precalibrated pore size demonstrated that the inhibitory activity was associated with a less than 10,000 mol wt fraction; 3 micrograms protein/assay of this fraction resulted in 50% inhibition (IC50) of maximal hCG stimulation. The inhibitory activity also passed through an Amicon YM-2 membrane (mol wt retention, 1,000), but not through an Amicon YC-05 membrane (mol wt retention, 500). An IC50 of about 0.01 microgram protein/assay was found for both the 500-1,000 and the 1,000-5,000 mol wt fractions. NaF or forskolin-stimulated adenylyl cyclase activity was not altered by unfractionated hFF or by the 500-10,000 mol wt subfractions, suggesting that inhibition was limited to LH/hCG stimulation. Further analysis of the effects of low mol wt fraction on hCG stimulation of adenylyl cyclase indicated that enzyme inhibition was not accompanied by a shift in the hCG concentration required for half-maximal stimulation (the apparent activation constant) compared to dose-response curves obtained in the absence of added fraction. Equilibrium binding studies showed that [125I]hCG interaction with luteal membranes was significantly inhibited by hFF; 7 micrograms protein/assay of the less than 10,000 mol wt fraction reduced specific binding by 60%. Moreover, kinetic analysis carried out in the absence or presence of a fixed amount of low mol wt fractions revealed a competitive type of binding inhibition. Our data demonstrate that a nonsteroidal component(s) of hFF has a direct inhibitory effect on LH/hCG-responsive luteal adenylyl cyclase and that the inhibitor(s) exerts its actions through a mechanism involving competition with LH/hCG for the same binding sites.

Opposite effects of ethanol on the activation of adenylyl cyclase in human corpus luteum membranes.

The influence of an acute exposure to ethanol on adenylyl cyclase activity in membrane fractions prepared from human corpus luteum was investigated. Ethanol up to a concentration of 5% (v/v) was without effect on basal luteal adenylyl cyclase activity, but markedly potentiated stimulation of NaF and hCG in a dose-dependent manner. In contrast, ethanol progressively inhibited forskolin stimulation at the same range of ethanol concentrations. Maximal NaF and hCG responsiveness of adenylyl cyclase activity was observed at 5% ethanol and reached values 80% and 100% higher than controls without ethanol, respectively. However, at the same ethanol concentration, forskolin-stimulated enzymatic activity was reduced by 40% relative to controls. Equilibrium binding studies involving [125I]hCG interaction with luteal membranes in the presence of the concentration of ethanol showing maximal hCG responsiveness indicated that ethanol slightly affected (15% increase) the hCG binding compared to controls, without any appreciable change on the Kd for the hormone. This minor effect of ethanol on gonadotropin binding sites contrasted greatly with the extent at which ethanol maximally potentiated the gonadotropin-stimulated adenylyl cyclase. GTP was found to be less effective than GMP-P(NH)P in sustaining ethanol potentiation, suggesting that ethanol is unlikely to act by inhibiting GTPase activity. These data indicate that the acute effects of ethanol inhibit forskolin-stimulated adenylyl cyclase at concentrations potentiating stimulatory effects of NaF and of hCG, and that the synergistic interaction of ethanol and gonadotropin stimulation of adenylyl cyclase is, at least in part, due to an increase in the functional coupling of the occupied hCG-receptor complex with the components of the enzyme system.

Effects of a long-acting LHRH agonist preparation on plasma gonadotropin and prolactin levels in castrated male rats and on the release of prolactin from ectopic pituitaries.

We have examined the effects of a single subcutaneous injection of an LHRH agonist, D-Trp-6-LHRH, in biodegradable microcapsules of poly(DL-lactide-co-glycolide) on plasma gonadotropin and prolactin (PRL) levels in castrated and in castrated-hypophysectomized-pituitary grafted (CAST-APX-GRAFT) male rats. The results were compared to the effects of daily injections of the same LHRH agonist dissolved in saline. In castrated rats, there were no significant alterations in plasma LH or PRL levels during the 10 days following the injection of LHRH agonist microcapsules, while FSH levels were generally reduced. In castrated males given daily injections of 6 micrograms of LHRH agonist in saline, plasma LH levels were significantly reduced while plasma PRL levels were not changed. In CAST-APX-GRAFT rats, both D-Trp-6-LHRH microcapsules and daily LHRH agonist injections appeared to increase plasma PRL levels. The pattern of changes in PRL release in both groups was similar, with levels on day 6 being significantly higher than those measured on days 1, 3 and 10 after onset of treatment. As expected, LH and FSH levels in these animals were extremely low. Immunoreactive D-Trp-6-LHRH was consistently detectable in the plasma of CAST-APX-GRAFT animals after microcapsule administration, whereas in animals given daily injections of this agonist in saline, its plasma concentrations were often below the detectability limit of the employed assay. These findings suggest that the LHRH agonist, D-Trp-6-LHRH, is capable of causing a short term stimulation of PRL release from ectopic pituitaries. Elevation of plasma LH levels is apparently not required for this effect.

Evidence that hormone receptors couple and activate a common signal transducer adenylyl cyclase in corpus luteum.

OBJECTIVES: To investigate whether membrane-bound hormone receptors in corpus luteum (CL) couple to one common adenylyl cyclase (AC) or whether each receptor is coupled to its own AC. DESIGN: Plasma membranes from rat CL were used to assess the coupling of hormone receptors to AC under conditions allowing full expression of the enzyme system. The response to hCG, prostaglandin E2 alpha (PGE2), and isoproterenol were analyzed. MAIN OUTCOME MEASURE: Adenylyl cyclase activity was monitored by the direct conversion of [infinity-32P]ATP into [32P]cyclic AMP. Results were expressed as pmol/min per mg membrane protein. RESULTS: Addition of hCG (200 mIU), PGE2 (100 microM), and isoproterenol(100 microM) in the presence of a saturating (100 microM) concentration of guanine nucleotide resulted in a marked stimulation of the enzyme compared with controls, reaching 552 +/- 28, 537 +/- 42, and 558 +/- 32 (mean +/- SEM), respectively. Addition of hormones in pairs or all three together did not result in an additive response of the individual effects. Thus, in the presence of the three hormones together, AC stimulation was 582 +/- 41 (n = 5), a value that was not significantly different from the stimulation observed with each hormone alone. CONCLUSION: These data indicate that luteal membranes contain a single AC system. Therefore, hormone receptors couple and activate a common signal transducer AC in luteal membranes.

The effect of bromocriptine on the motility of human spermatozoa and its capacity to penetrate the cervical mucus.

We investigated the direct effects of bromocriptine on the motility of the human ejaculated spermatozoa and its capacity to penetrate the cervical mucus (CM) in vitro. Washed sperm were incubated with a wide range of bromocriptine concentrations (0.005 to 5 mM). Progressive and total motility was evaluated after 15, 30, 60, and 180 minutes. No reduction of motility was observed at any concentration tested. Similar results were observed with semen samples. Failure to alter sperm motility was evident in samples with either good or reduced motility. Also, CM penetration as measured after 90 minutes by Penetrak assay (Serono Diagnostics, Randolph, MA) was not impaired by a concentration as high as 5 mM bromocriptine. We conclude that bromocriptine in a wide range of concentrations does not inhibit sperm motility nor does it impair the capability of the sperm to penetrate the CM. These data support the therapeutic use of daily vaginal bromocriptine for the treatment of infertility.

Acrosome reaction of human spermatozoa in zona-free hamster egg penetration test.

The acrosomal status of human sperm during preparation for the process of zona-free hamster egg penetration test (ZFHEPT) was determined. The incidence of acrosome reaction (AR), as assessed by triple-stain technique, was significantly increased after 24 hours of incubation at 4 degrees C in TES-Tris (TEST)-yolk buffer, but the absolute values were relatively low (20% or less). Sperm from fertile donors and infertile patients with normal or abnormal semen analysis displayed similar capacity to undergo the AR in vitro. Although a positive correlation was found between the incidence of AR and the score of ZFHEPT, a remarkable individual variation was noted. The incidence of AR in freely swimming human sperm does not accurately reflect the fertilizing ability of the sperm.

Intrauterine inseminations with washed human spermatozoa does not induce formation of antisperm antibodies.

In this study we investigated the possible development of serum antisperm antibodies in women receiving repeated IUI. Patients acted as its own control and were evaluated before and after various (1 to 15) IUI cycles using three different assays for antisperm antibodies. It was found that only 2 out of 41 women developed antisperm antibodies. We concluded that exposure of the upper reproductive tract to washed spermatozoa during repeated IUI with partners' sperm does not significantly stimulate the appearance of serum antisperm antibodies.

Enzymatic amplification of specific deoxyribonucleic acid sequences from single cells: evaluation of a simplified and rapid method for use in preimplantation genetic diagnosis.

OBJECTIVE: To develop a simplified polymerase chain reaction (PCR) protocol on single cells for the purpose of preimplantation genetic diagnosis. Also to evaluate a new thermal cycler, RoboCycler 40 (Stratagene, La Jolla, CA), for reducing the time to complete PCR amplification. DESIGN: PCR amplification without DNA purification or reamplification of a 149 base pair (bp) segment of the human Y chromosome was used as a model. The assay was tested in human fetal cells, single lymphocytes and single human blastomeres. RESULTS: Amplification of the 149 bp segment using fetal cells was 100% correct. Results on single lymphocytes were concordant in all but one of the 15 male cases. However, 2 of the 25 female cases were identified as male suggesting the occurrence of DNA contamination. Analysis of 61 blastomeres were concordant in 57 cases (93%); results for male blastomeres showed 12% of false negatives. No false positives were detected for female cells. Amplification using the simplified PCR protocol in combination with the RoboCycler was completed in 2 hours. CONCLUSION: These data show that this PCR assay performed directly, without DNA extraction or purification and without re-amplification is a practical and effective approach for amplification of specific DNA sequences in single cells. Furthermore, the simplified PCR protocol significantly reduced the time to complete DNA amplification. The reduced time is expected to facilitate the management of a routine program for preimplantation genetic diagnosis.

Successful gamete intrafallopian transfer following failed artificial insemination by donor: evidence for a defect in gamete transport?

Gamete intrafallopian transfer (GIFT) was offered as an alternative treatment to 48 women who failed to conceive after artificial insemination with donor semen (AID) in numerous attempts (9 to 24 cycles). The evaluation of these women showed no major cause of infertility as evidenced by normal endocrine, cervical, uterine, and tubal factor studies. Their partners were either azoospermic or severely oligoasthenospermic. During the GIFT cycle, follicular development was induced with (1) clomiphene citrate (days 3 to 7) plus human menopausal gonadotropins (hMG) from day 6 on or (2) human follicle-stimulating hormone (days 3 to 4) plus hMG (day 5 on), until ultrasound revealed 2 follicles 16 mm and serum estradiol (E2) was greater than 700 pg/ml. Human chorionic gonadotropin (hCG) 10,000 IU was administered, and 36 hours later follicular aspiration was performed. One to three oocytes and 100,000 motile sperm were transferred to each fallopian tube through the fimbria via laparoscopy or minilaparotomy. Twenty-seven clinical pregnancies were achieved (56%) per GIFT cycle. Eight miscarriages occurred during the first trimester (29% of all pregnancies), whereas no ectopic pregnancies were observed. These data conclusively show the value of the GIFT procedure in the treatment of cases with failed AID.

Penetration of zona-free hamster oocytes using human sperm aspirated from the epididymis of men with congenital absence of the vas deferens: comparison with human in vitro fertilization.

OBJECTIVES: To assess the ability of sperm aspirated from the epididymis of men with congenital absence of the vas deferens to penetrate zona-free hamster oocytes. To directly compare the performance of human epididymal sperm in the zona-free hamster oocyte sperm penetration assay (SPA) with the results of human in vitro fertilization (IVF). DESIGN: Sperm penetration assay was carried out with epididymal sperm retrieved microsurgically, and with ejaculated sperm obtained from fertile donors (internal controls). For direct comparison, SPA was performed with the same epididymal sperm sample used for IVF. PATIENTS, PARTICIPANTS: Men with congenital absence of the vas deferens undergoing sperm aspiration as part of their infertility treatment and control donors who provided ejaculated sperm. RESULTS: Epididymal sperm penetrated SPA with a score of 0% to 30%. The SPA scores for internal controls using ejaculated sperm was 30% to 71%. Linear regression analysis of the association between penetration scores in SPA and fertilization rate in IVF indicated a positive correlation that was highly significative. CONCLUSIONS: These findings using SPA confirm previous reports on the fertilizing potential of human epididymal sperm and its ability to produce normal pregnancies. The good correlation between SPA and human IVF using epididymal sperm suggest that SPA is an excellent bioassay to test laboratory experimental conditions for improving fertilizing capacity of human epididymal sperm.

Failed oocyte retrieval after lack of human chorionic gonadotropin administration in assisted reproductive technology.

OBJECTIVE: To document the absence of oocytes in follicular aspirates in women who, during controlled ovarian stimulation with gonadotropin-releasing hormone agonist (GnRH-a) and menotropins, fail to receive human chorionic gonadotropin (hCG) administration. DESIGN: Retrospective analysis of clinical laboratory data. SETTING: Multicentric. PATIENTS: Five women undergoing controlled ovarian hyperstimulation with GnRH-a and menotropins for programs of assisted reproductive technologies. RESULTS: The documented absence of an hCG injection produced "empty follicles" at transvaginal guided aspiration, despite numerous follicular lavages and aspiration of peritoneal fluid. The lack of oocytes and granulosa-cumulus complex in the follicular fluid was reverted in other cycles in the same patients when hCG was properly administered. CONCLUSIONS: (1) This study emphasizes the importance of proper patients' and nurses' instructions for preparation of hCG injections and proper mixture of vehicle and powder before follicular aspiration. (2) In the absence of cumulus-corona-oocyte complex at aspiration, measure serum beta-hCG to ascertain whether hCG injection was administered or not. (3) Routine preoperative beta-hCG levels may be helpful to avoid unnecessary surgeries.

The predictive value of a single beta human chorionic gonadotropin in pregnancies achieved by assisted reproductive technology.

OBJECTIVE: To investigate whether a single serum beta-hCG in pregnancies achieved by assisted reproductive technologies (ART) can accurately predict pregnancy viability and, in viable pregnancies, multiple gestation. DESIGN: Four hundred sixty-one consecutive successful ART pregnancies were studied retrospectively. Seventy-one of the 461 patients were excluded because their beta-hCG was either drawn on the incorrect day or outside our facility. Three hundred ninety subjects had a serum beta-hCG drawn 14 days after ET or 16 days after gamete transfer. The beta-hCG samples were analyzed by immunoradiometric assay based on the Third International Reference Standard (IRS) (First International Reference Preparation (IRP)). Pregnancy status was followed, at minimum, through the first trimester. RESULTS: One hundred fifty (38%) of the 390 were found to be nonviable, resulting in spontaneous abortion (n = 38, 10%), ectopic pregnancy (n = 27, 6%), or biochemical pregnancies (n = 85, 22%). A statistically significant difference by the Scheffe F-test was found between the mean beta-hCG value of the nonviable (115 mIU/mL) (conversion factor to SI unit, 1.00) and viable (428 mIU/mL) pregnancies. The positive predictive value of a single beta-hCG > 100 mIU/mL in distinguishing viable from nonviable pregnancies was 0.83 (sensitivity 91%, specificity 71%). Of the 240 viable pregnancies, 74 (32%) were multiple gestations (57 twins, 14 triplets, and 3 quadruplets). The mean beta-hCG of the singleton pregnancies (266 mIU/mL) was significantly different from that of the multiple gestations (792 mIU/mL). The positive predictive value of a single serum beta-hCG < or = 400 mIU/mL in distinguishing singleton from multiple gestations was 0.92 (sensitivity 86%, specificity 82%). CONCLUSION: A single early serum beta-hCG may be used in ART pregnancies to predict which pregnancies will continue beyond the first trimester and to identify multiple gestations. Early reassuring tests may reduce anxiety.